Nanoscale regulation of Ca2+ dependent phase transitions and real-time dynamics of SAP97/hDLG

Synapse associated protein-97/Human Disk Large (SAP97/hDLG) is a conserved, alternatively spliced, modular, scaffolding protein critical in regulating the molecular organization of cell-cell junctions in vertebrates. We confirm that the molecular determinants of first order phase transition of SAP97/hDLG is controlled by morpho-functional changes in its nanoscale organization. Furthermore, the nanoscale molecular signatures of these signalling islands and phase transitions are altered in response to changes in cytosolic Ca2+. Additionally, exchange kinetics of alternatively spliced isoforms of the intrinsically disordered region in SAP97/hDLG C-terminus shows differential sensitivities to Ca2+ bound Calmodulin, affirming that the molecular signatures of local phase transitions of SAP97/hDLG depends on their nanoscale heterogeneity and compositionality of isoforms.

Further, the work examines the effect of [Ca2+]i on the organization and phase transition order of SAP97. Authors dissected various features related to different regions of the molecules and also isoforms.
Evidently, authors have performed several clever experiments and utilized various tools to quantitate the role of Ca2+. And admittedly, some of those experiments are beyond my expertise. I expect that the soundness of the data in experiments such as microscopy would be judged by other reviewer(s).
In my assessment, though the manuscript contains substantial quantity of data, proper communication is lacking. For example, the second result, which has been titled in the manuscript as "Modulation of binding of SAP97/hDLG to Ca2+ alters dynamics of first order phase transitions", wrongly implies that SAP97 binds Ca2+. I am not sure if SAP97 has Ca2+ binding motif(s). Ca2+ binding is considered highly specific, and binds at a geometry or motif. This needs to be clarified or corrected, since one gets similar impression at multiple instances.
Another statement: "To confirm the CaM independent Ca2+ interaction of I2 isoform, the cells were treated…. (para 1 on page 13)" (it implies to me that I2 form binds Ca2+). In my knowledge MAGuKs do not bind Ca2+ directly (please correct me if it is wrong!), though being important scaffold protein, they play important role in neuronal plasticity and memory by interacting with a number of proteins in synapse.
CaM binding to SAP97 is Ca2+ dependent, and obviously, a perturbation of intracellular Ca2+ affects the organization of SAP97 via its interaction with CaM. Thus, a defined dynamical relationship exists between intracellular Ca2+ levels and the organization of SAP97.
The manuscript certainly contains interesting results. Authors have cleverly demonstrated roles/dynamics of intrinsically unstructured regions and spliced variants with respect to the change in the [Ca2+]i. However, the fact that it is mediated by CaM should be reflected, even in the subtitle of the manuscript (if it is so).
The language of the manuscript needs to be corrected for basic grammar, typos and proper expression of units.
Reviewer #3 (Remarks to the Author): Rajeen et al., is describing functional and nanostructural characteristics of a scaffolding platform SAP97/hDLG in Neuro-2a cells. By using a combination of techniques including dSTORM nanoscopy and FRAP they elegantly characterize morphological properties of these hubs at the nanoscale level and link them to organizational phase transitions (of the first order). Notably, they use the number of molecules inside the cluster and pool of free monomers that can be extracted from dSTORM imaging, to evaluate the free energy change (bulk and surface). By perturbing the intracellular calcium, the authors show the role of this ion binding in modulating the phase transition inside the SAP97/hDLG nanoclusters.
Phase transition in molecular aggregates and membrane-less molecular condensates is currently a hot topic across different biological disciplines. Therefore I am confident that the results and the elegant approach presented in this paper will be of interest to the broad scientific community, especially that SAP97/hDLG hub has importance in many cells to cell adhesion systems. I do not have major remarks, but rather minor suggestions.
-First, it does not transpire from the current manuscript what is the core of the discussed phase transition; structural phase transitions, but what actually could be happening there, e.g. molecules are more packed but is there chemical evidence for that?. A less informed reader would benefit from a more basic introduction to this problem, which would greatly facilitate absorption of the presented methodology, data, and their interpretation.
-The result section is a bit technical. For clarity, maybe the authors would find a way to move some part of the more detailed description of the procedure to the Material and Methods section. This would facilitate understanding of the actual results and their significance.
-Authors showed that blocking the calcium sensor interaction with SEP97/hDLG leads to an increase in the molecules inside the cluster, but only a moderate increase in the cluster area. Could authors discuss whether this, at least in part, could be explained by the resolution limit of their microscope?
This question relates to wherever the changes in the intensity and the area of the cluster are discussed.
-Authors use Kolmogorov-Smirnov test. Why? The data wasn't normally distributed in general?

Response to Reviewers
We are grateful to the editor and the reviewers for evaluating the manuscript positively and for their insightful comments to strengthen the manuscript. We have performed all the additional analyses, experiments to answer the concerns raised by the reviewers. We have added one additional figure to the main manuscript and added five supplementary figures to support the experiments performed for the revised manuscript in addition to alterations in the main text to address the queries raised by the reviewer. We believe that addressing these comments have enhanced the manuscript in its scientific content, technical execution, and its overall quality.
A summary of the additional experiments and modifications for the revised version of the manuscript is listed below. The corresponding alterations in the revised manuscript text are highlighted in yellow.
1. In response to the query from reviewer 1 we have added in vitro experiments in the revised manuscript to confirm that phase transition of SAP97/hDLG is homotypic and is an intrinsic property of isoforms and that calcium is a pre-requisite for heterotypic condensation of SAP97/hDLG and Calmodulin. This query from reviewer 1 also answers the minor comment from reviewer 3 2. In response to the query from reviewer 1 we have investigated the SAP97/hDLG distribution in Neurons. SAP97/hDLG; both ectopically expressed (PALM) and endogenous (STORM) form nanoscale condensates in polarized cells such as neurons. Majority of SAP97/hDLG condensates in dendritic spines were observed at the periphery of the postsynaptic density. The in vitro and in vivo data confirm that condensation of SAP97/hDLG is influenced by the combination and/or spatial availability of isoforms. 3. We have also addressed the comments from reviewer 2 and 3 to reduce the conceptual gaps and to improve the readability of paper giving it a broader perspective. We have rectified the typos, simplified the introduction, results, and discussion and edited the language of paper to address the comments. Over the last decade, our understanding of the formation, maintenance and elimination of molecular clusters is getting re-evaluated vigorously. In contrast to a controlled environment that we see in solutions, the natural environment in cells offers a different kind of challenge where these molecules transiently interact with more than one molecule at any instant. Such multicomponent interaction offers a very fine scale regulation of membrane associated complexes due to association and dissociation of transmembrane molecules, scaffolds, and other signalling molecules. The research from our group and others have shown that many of these regulatory nanodomains are below the limit of diffraction and are formed at a spatial scale of 10-200 nm transiently at millisecond-minute time scales in cells [1][2][3][4][5] . Recent evidence confirms that molecules involved in these transient domains also participate in liquid-liquid phase separation (LLPS) in vitro [6][7][8] . Additionally, a long line of research using techniques such as Fluorescent Correlation Spectroscopy, Fluorescence Recovery after photobleaching and Single Molecule Tracking experiments point out that molecules involved in the formation of nano to micron sized domains also have the potential to have altered kinetics of exchange 1,9,10 . Such differing kinetics arise as a response to cellular environment or modification that happens post translationally, or as a consequence of multicomponent system within a cell. As of now, there are very few studies that offers insights into the nanoscale regulation of many of these clusters in their natural environment. A recent observation identified the potential of advanced microscopy to understand this question directly from cells 11,12 .To the best of our knowledge, super resolution microscopy is one of the few techniques that can be used to observe the heterogeneity of molecular content of nanoscale structures in a multicomponent system such as a cell. Thus, correlation of super resolution microscopy with FRAP provides a detailed information on these complexes at nanoscale spatial and millisecond temporal precision. Advanced microscopy techniques in combination with novel analysis paradigms provides insights into this regulation, which makes the experimental paradigm suitable and relevant for this study. Additionally, we agree with the reviewer on the need to evaluate if liquid-liquid phase transitions are an intrinsic property of SAP97/hDLG and how these condensates respond to intracellular alteration resulting from an increased elevation in calcium. To address this, we have performed in vitro phase transition studies and confirmed that the isoforms of SAP97/hDLG can transition into phase separated condensates and can recruit Calmodulin into them in a calcium dependent manner. These experiments strengthen the results observed using super resolution microscopy and FRAP, allowing us to probe deeper into submicron scale regulation of these clusters, that is not easily accessible by in vitro phase separation experiments.
We thank the reviewer for the queries and strongly believe that the revised paper and the additional experiments prove that there is an isoform specific difference in the phase transition of SAP97/hDLG, which contributes to the regulation of its nanoscale condensates. Since hDlg is easy to be purified, they need to validate the phase transition ability of hDlg using purified proteins in vitro.
We appreciate this relevant concern of the reviewer. The inferences on the nanoscale regulation of SAP97/hDLG were based on the figures, Figure1, 2 and 3 11,13,14 . Figure 1A is a representative example of several cells that was used to compare and explain the general framework for acquisition and analysis. This comprehensive analysis pipeline allowed us to reach the conclusion as presented in Figure1B-E in control conditions and Figure 2 between different conditions of modulating the intracellular calcium levels as well as the interaction of calcium with Calmodulin. These methods are comparable to what has been reported previously to explain phase transitions using different forms of microscopy 11,14 . Figure 3 uses rank order analysis, which provides a holistic insight into morpho-Response to reviewer Figure2: Isoform specific difference in the nucleation of C-terminus of SAP97/hDLG: Top and bottom panels indicate the phase separation of C terminal isoforms, namely SH3-I2-GUK::GFP and SH3-I3-GUK::GFP, respectively in response to their increasing concentration at 20% PEG. SH3-I2-GUK::GFP nucleation occurs at a concentration higher than SH3-I3-GUK::GFP, indicating that spontaneous phase transition of I3 isoform of SAP97/hDLG occurs at a lower concentration than that of I2 isoform. Scale bar indicates 5µm.
The reviewer is correct that the Figure S4A does not present typical round puncta-like structures formed via phase transition. As of now there is growing evidence of LLPS for several key molecules critical in polarized cell junctions and at neuronal synapses. In comparison to other molecules involved in the control of translation machinery these molecules also form microscale phase separated domains in vitro. However, majority of these molecules involved at polarized cell junctions do not form such large clusters in their natural environment 6,7 . Expression of these molecules in their natural environment result in patterns varying from soluble to punctate structures with diverse size ranges when observed by diffraction limited light microscopy. Interestingly, the size of an average excitatory postsynaptic density falls between 300-600nm, within which these molecules are clustered into domains of 10-200nm in diameter 1,[5][6][7]11 . The subsynaptic clusters are not resolvable by conventional microscopy paradigms. The results that we present are consistent with this model. The complex multicomponent interactions present within cells may restrict the formation of large micron scale clusters in contrast to a single component system or expressing a truncated protein may allow selective interaction with reduced number or moieties, resulting in an altered macromolecular equilibrium. Response to reviewer Figure 5: Concentration dependent recruitment of Calmodulin to SAP97/hDLG condensates in presence of calcium: 1µM SH3-I2-GUK::GFP and SH3-I3-GUK::GFP and 2mM CaCl2 were co-incubated with increasing concentrations of CaM::mCherry from 100nm to 2µM. CaM::mCherry was recruited into SAP97/hDLG condensates when it was 2µM for SH3-I2-GUK::GFP and 1µM for SH3-I3-GUK::GFP, respectively. This shows that CaM requires both SAP97/hDLG and calcium for recruitment into liquid liquid phase separated domains of SA97. Additionally, the co-condensation of CaM with I3 was observed at a lower concertation of CaM, as compared to I2 isoform. Scale bar indicates 5µm.

The cell model that the authors chose is strange. hDLG most likely form clusters in cellcell contact or polarized cells. So, it is strange to image an individual, unpolarized Neuro-2a cells.
Unpolarized Neuro-2a cells were adopted for this study for the following reasons: 1, To show that the phase transition of SAP97/hDLG is an intrinsic property. 2, In Neuro-2a cells, I2 isoform of SAP97/hDLG contributes to more than 97% of expressed mRNAs, while in both hippocampal and cortical neurons I2 and I3 were present in comparable levels.
One of the hypotheses in our paper is that the local compositionality of isoforms could have a direct consequence on the nanoscale condensation of SAP97/hDLG, as observed by light microscopy.
Considering the phase transition of SAP97/hDLG to be dependent on the properties of isoforms, neurons and Neuro-2a cells present different combinations of its C terminal spliced variants. Consistent with previous reports 26,27 , by ectopic expression of SAP97/hDLG/hDlg isoform (Response to reviewer Figure 6A) as well as using an antibody (Response to reviewer Figure 6B) that labels all reported isoforms of SAP97/hDLG we observed nanoscale condensation of SAP97/hDLG in dendritic shafts, morphologically characterized spines and in the proximity of postsynaptic density (Response to reviewer Figure 6A, B). Additionally, multicolour super resolution microscopy of the postsynaptic density protein PSD95 and SAP97/hDLG indicated that the majority of the condensates are formed peripheral to the postsynaptic density (Response to reviewer Figure  6B). In single synapses where both PSD95 and SAP97/hDLG were colabelled we found that they overlapped at their periphery and that their centre of the distribution was within 500nm (Response to reviewer Figure 6B). Using an antibody that labels all reported isoforms of SAP97/hDLG, we I3 and I2 isoforms and untransfected Neuro-2a cells. N for each condition = 8-11cells, Plots represent mean ± s.e.m. * = p<0.05, ** = p<0.01, **** = p<0.0001.
In summary the suggestions from reviewer allowed us to 1) Confirm that phase transition of SAP97/hDLG is homotypic and is an intrinsic property of isoforms 2) Calcium is a prerequisite for heterotypic condensation of SAP97/hDLG and Calmodulin 3) SAP97/hDLG; both ectopically expressed (PALM) and endogenous (STORM) form nanoscale condensates in polarized cells like neurons. 4) Majority of SAP97/hDLG condensates in dendritic spines were observed at the periphery of the postsynaptic density 5) The condensation of SAP97/hDLG is influenced by the combination and/or spatial availability of isoforms.

Reviewer #2 (Remarks to the Author):
The manuscript deals with the identification of phase order transition in the organization of SAP97/hDLG in vivo, which, owing to its size, has to be demonstrated by super resolution spectroscopy. The conclusion drawn was that SAP97/hDLG is largely organized on nanoscale level. Further, the work examines the effect of [Ca 2+ ] on the organization and phase transition order of SAP97/hDLG. Authors dissected various features related to different regions of the molecules and isoforms.
We thank the reviewer for comprehending the crux of the message of the paper, and for appreciating the relevance of the results and scope of the work. We feel that the suggestions from the reviewer have greatly improved the conceptual gaps and readability of the paper, giving it a broader perspective.

CaM binding to SAP97/hDLG is Ca 2+ dependent, and obviously, a perturbation of intracellular Ca 2+ affects the organization of SAP97/hDLG via its interaction with CaM. Thus, a defined dynamical relationship exists between intracellular Ca 2+ levels and the organization of SAP97/hDLG.
The reviewer comment is very valid 16,17 . Our experiments in response to questions from reviewer 1 gives a direct proof of relationship between intracellular Ca 2+ levels, CaM and the organization of SAP97/hDLG. However, in a cellular environment, this organization is fine-tuned at a molecular scale. We have emphasized this again in the discussion of the revised manuscript.

The manuscript certainly contains interesting results. Authors have cleverly demonstrated roles/dynamics of intrinsically unstructured regions and spliced variants with respect to the change in the [Ca 2+ ]i. However, the fact that it is mediated by CaM should be reflected, even in the subtitle of the manuscript (if it is so).
We thank the reviewer for this relevant comment. We have emphasized explicitly that alteration in the dynamics of SAP97/hDLG spliced variants with respect to the change in [Ca 2+ ] is mediated by Calmodulin both in the introduction and discussion section of the revised manuscript.

The language of the manuscript needs to be corrected for basic grammar, typos and proper expression of units.
We thank the reviewer for the comment. The modified manuscript has been proofread for the clarity of scientific work and readability by three native speakers working in microscopy and cell biology We believe that the overall quality of the manuscript has been enhanced by these modifications.

Rajeev et al., is describing functional and nanostructural characteristics of a scaffolding platform SAP97/hDLG in Neuro-2a cells. By using a combination of techniques including dSTORM nanoscopy and FRAP they elegantly characterize morphological properties of these hubs at the nanoscale level and link them to organizational phase transitions (of the first order). Notably, they use the number of molecules inside the cluster and pool of free monomers that can be extracted from dSTORM imaging, to evaluate the free energy change (bulk and surface). By perturbing the intracellular calcium, the authors show the role of this ion binding in modulating the phase transition inside the SAP97/hDLG nanoclusters.
We are grateful to the reviewer for identifying the core message of the paper, and for appreciating the quality of data and the outcome of results. The comments from the reviewer have helped us to make the paper suited to a broader range of readers with a streamlined flow of results.
Phase transition in molecular aggregates and membrane-less molecular condensates is currently a hot topic across different biological disciplines. Therefore, I am confident that the results and the elegant approach presented in this paper will be of interest to the broad scientific community, especially that SAP97/hDLG hub has importance in many cells to cell adhesion systems.
We thank the reviewer for the kind opinion 11,14 . We have added additional in vitro data and have extended the work to compare molecular fingerprints of this phase transition to varying compositionality of SAP97/hDLG isoforms. We believe that these experiments complement the overall message of the paper.

I do not have major remarks, but rather minor suggestions. -First, it does not transpire from the current manuscript what is the core of the discussed phase transition; structural phase transitions, but what actually could be happening there, e.g. molecules are more packed but is there chemical evidence for that?. A less informed reader would benefit from a more basic introduction to this problem, which would greatly facilitate absorption of the presented methodology, data, and their interpretation.
This is a relevant comment from the reviewer which has been addressed in the revised manuscript. The introduction has been edited to include the attention of a diverse audience. The invitro assays that we have performed for phase transitions provide chemical evidence for packing. We request the attention of the reviewer to Response to reviewer figures 1-5 which are also included in the results section of the revised manuscript. -Authors showed that blocking the calcium sensor interaction with SEP97/hDLG leads to an increase in the molecules inside the cluster, but only a moderate increase in the cluster area. Could authors discuss whether this, at least in part, could be explained by the resolution limit of their microscope? This question relates to wherever the changes in the intensity and the area of the cluster are discussed.
The smallest molecular domains that can be obtained by STORM is determined by the localization precision of the microscope 30,31 . This results in an inherent limitation to quantify the domains which are below the resolution limit. However, our results on the molecular domains are well above this pointing accuracy. The changes in the area were also significant in the analysis as indicated by up valuesv. The interesting aspect was that the scaling in area was not directly proportional to the scaling in the molecular content as indicated by the number of SAP97/hDLG molecules detected in these molecular domains. We agree with the reviewer that resolution influences the quantification of microscopy images, but the data have been acquired with the best spatial resolution possible within the scope of the article, which will not change the outcome of the result. We value the reviewer comment and have added further clarification in the discussion section of the revised manuscript.

-Authors use Kolmogorov-Smirnov test. Why? The data wasn't normally distributed in general.
We thank the reviewer for this relevant question on the statistical analysis of the data. Every result was analysed for the normality of the distribution. The data was found to have a non-normal distribution. For this reason, we have chosen Kolmogorov-Smirnov test. We have specified this point in the discussion of the revised manuscript.
In the revised manuscript, the authors provided evidence showing that SH3-HOOK-GUK of SAP97 I2/I3 isoforms underwent phase separation in vitro, and CaM could be recruited into SAP97 condensates to alter their internal dynamics in the presence of Calcium. They further showed that SAP97 form nanoscale condensates in neurons, and the condensation is influenced by the compositionality of SAP97 isoforms. My concerns have been adequately addressed. The revised paper carefully describes an interesting and important study that should be of general interest. I recommend that it is accepted without further revision.
Reviewer #2 (Remarks to the Author): I went through the revised version, where the authors have taken care of most of my concerns. My major concern was about the notion that indicates Ca2+ binding to SAP97. It was a technically and scientifically wrong conception inferring that SAP97 is a Ca2+ binding protein. The authors have toned that down in their revision. However, I see some points still in the manuscript, which I find quite uncomfortable to accept, unless proven scientifically correct (authors claim they are..) For example, "Since SAP97/hDLG senses Ca2+ through Calmodulin,…." SAP97 and CaM binding is strictly Ca2+ dependent as mapped earlier. An alteration in the intracellular Ca2+ level would affect this binding, and hence further downstream functions. This however, does not infer that SAP97 could sense Ca2+ (even in the presence of CaM).
Other statements from the manuscript: "Differential sensitivity of I2 and I3 SAP97/hDLG variants to cytosolic Ca2+ through CaM modulate their exchange kinetics.." "To confirm whether CaM modulates the Ca2+ dependent response of SAP97/hDLG,….." It is safer to avoid such conflicting statements which also indicate the same erroneous notion. CaM has many targets where the association is strictly Ca2+ dependent. Some of these targets also undergo structural changes once holo-CaM binds. Will such targets be categorized as calciumdependent or -sensitive that they sense Ca2+ through CaM (as per your method)?
I agree that a pharmacological treatment for elevating the intracellular Ca2+ levels in cell culture demonstrates the dynamic changes in the clusters of SAP97. This necessarily does not infer that SAP97 is Ca2+ sensitive unless proven in vitro. While the data are interesting, the interpretation by authors is not acceptable. After addressing different reviewers' comments, this manuscript has significantly improved. Authors performed new experiments and analyses to carefully address comments, remarks, and questions raised by reviewers. In this new shape, I found that the authors have gathered enough data to support their conclusions, as requested by the reviewers.
The phase transition in cellular condensate (nano, and microsized) is becoming a hot topic across different biological disciplines.
This manuscript presents very interesting insights concerning the formation of SAP97/hDLG condensates and their regulation by Ca2+ through HOOK-CaM. Notably, phase transitions were observed at multiple scales, from molecular to supramolecular.
New concepts, suit of technics including SMLM (PALM, dSTORM) and FRAP, and novel quantitative analyses developed by the authors will be of great interest to scientists other than neuroscience disciplines.
Notably, the authors developed a very interesting and original way to extract thermodynamic parameters involved in cellular condensate transitioning from super-resolved images.
Therefore, I am convinced that this manuscript will be of great interest to the broad scientific public interested in the mechanism involved in the self-organization of molecular condensates.

Response to Reviewers
We are grateful to the editor and the reviewers for evaluating the manuscript positively and for their insightful comments to strengthen the manuscript. It has been a delightful experience to address the queries raised by the editor and reviewers, that has greatly improved the final version of the manuscript.
- In the revised manuscript, the authors provided evidence showing that SH3-HOOK-GUK of SAP97 I2/I3 isoforms underwent phase separation in vitro, and CaM could be recruited into SAP97 condensates to alter their internal dynamics in the presence of Calcium. They further showed that SAP97 form nanoscale condensates in neurons, and the condensation is influenced by the compositionality of SAP97 isoforms. My concerns have been adequately addressed. The revised paper carefully describes an interesting and important study that should be of general interest. I recommend that it is accepted without further revision.
We are grateful to the reviewer for the encouraging comments and for appreciating our efforts in the revised manuscript to address the reviewer concerns. We believe that the suggestions from the reviewer have significantly improved the technical and scientific perspectives of the manuscript.

Reviewer #2 (Remarks to the Author):
I went through the revised version, where the authors have taken care of most of my concerns. My major concern was about the notion that indicates Ca 2+ binding to SAP97. It was a technically and scientifically wrong conception inferring that SAP97 is a Ca2+ binding protein.
The authors have toned that down in their revision. However, I see some points still in the manuscript, which I find quite uncomfortable to accept, unless proven scientifically correct (authors claim they are..) For example, "Since SAP97/hDLG senses Ca 2+ through Calmodulin,…." We thank the reviewer for comprehending the crux of the message of the paper, and for appreciating the relevance of the results and scope of the work. We feel that the suggestions from the reviewer have improved the conceptual gaps and readability of the paper, giving it a broader perspective. We are grateful to the reviewer for the relevant comments. The reviewer is correct that SAP97/hDLG to the best of our knowledge do not have any motifs to directly bind Ca 2+1-4 . SAP97/hDLG binds to Ca 2+ bound Calmodulin. In the first revision itself, we had taken cognizance of the reviewer comments and requested three of our colleagues to read the paper to check for contextual lack of clarity raised by the reviewer. We apologize for any similar errors in the revised manuscript, which we have addressed thoroughly. 7 additional sentences have been corrected in the currently revised manuscript for addressing the reviewer concern including those given below.
"Since SAP97/hDLG senses Ca 2+ through Calmodulin, we confirmed the presence of its C-terminal spliced isoforms in the hippocampal and cortical regions of rodent brains as well as in heterologous cell lines" is changed to "Since SAP97/hDLG is reported to associate with [Ca 2+ .CaM], we confirmed the presence of its C-terminal spliced isoforms in the hippocampal and cortical regions of rodent brains as well as in heterologous cell lines." We have also added an additional sentence in the discussion that "Currently there is no experimental evidence to suggest that SAP97 can directly bind Ca 2+". SAP97 and CaM binding is strictly Ca 2+ dependent as mapped earlier. An alteration in the intracellular Ca 2+ level would affect this binding, and hence further downstream functions. This, however, does not infer that SAP97 could sense Ca 2+ (even in the presence of CaM).
We are in resonance with the reviewer comment. Sap97 does not sense or bind Ca 2+ directly, which has been clarified throughout the manuscript. Any sentence giving a similar message has been corrected accordingly.
Other statements from the manuscript: "Differential sensitivity of I2 and I3 SAP97/hDLG variants to cytosolic Ca 2+ through CaM modulate their exchange kinetics…." The statement has been altered as following in the revised manuscript, as per the suggestion from the reviewer. "Differential binding of I2 and I3 SAP97/hDLG variants to [Ca 2+ .CaM] modulate their exchange kinetics" "To confirm whether CaM modulates the Ca 2+ dependent response of SAP97/hDLG,….." The revised manuscript is altered as per the suggestion from the reviewer. The corrected statement reads as follows: "To confirm differential sensitivity of SAP97/hDLG to [Ca 2+ .CaM], we inhibited the… " It is safer to avoid such conflicting statements which also indicate the same erroneous notion. CaM has many targets where the association is strictly Ca 2+ dependent. Some of these targets also undergo structural changes once holo-CaM binds. Will such targets be categorized as calcium-dependent or -sensitive that they sense Ca 2+ through CaM (as per your method)?
We thank the reviewer for the suggestion and apologize for the confusion. The revised manuscript is corrected to clarify that SAP97/hDLG does not sense or bind Ca 2+ directly.
I agree that a pharmacological treatment for elevating the intracellular Ca 2+ levels in cell culture demonstrates the dynamic changes in the clusters of SAP97. This necessarily does not infer that SAP97 is Ca 2+ sensitive unless proven in vitro. While the data are interesting, the interpretation by authors is not acceptable. SAP97/hDLG does not sense or bind Ca 2+ directly, which has been clarified throughout the manuscript. The interpretation of the manuscript is in resonance with the reviewer's comments. We apologize for any mistake in conveying the message, which is corrected in the revised manuscript, as per the reviewer suggestion.

Some minor corrections:
Heading in Methods: not clear -The revised manuscript is altered for a better clarity of headings in Methods, as per the reviewer suggestion.

Protein expression, purification Expression-
The revised manuscript is altered with the corrected headings, as per the reviewer suggestion.
Inconsistency: Ca 2+ (in many places it is represented as calcium). The correct form in Ca 2+ (calcium ion, and not calcium)-The revised manuscript is corrected for this inconsistency, as per the reviewer suggestion.
Casual presentation of numbers: 10mM (should be 10 mM)-The revised manuscript is altered, as per the reviewer suggestion.

Reviewer #3 (Remarks to the Author):
After addressing different reviewers' comments, this manuscript has significantly improved. Authors performed new experiments and analyses to carefully address comments, remarks, and questions raised by reviewers. In this new shape, I found that the authors have gathered enough data to support their conclusions, as requested by the reviewers.

The phase transition in cellular condensate (nano and micro sized) is becoming a hot topic across different biological disciplines.
This manuscript presents very interesting insights concerning the formation of SAP97/hDLG condensates and their regulation by Ca 2+ through HOOK-CaM. Notably, phase transitions were observed at multiple scales, from molecular to supramolecular.
New concepts, suit of technics including SMLM (PALM, dSTORM) and FRAP, and novel quantitative analyses developed by the authors will be of great interest to scientists other than neuroscience disciplines.
Notably, the authors developed a very interesting and original way to extract thermodynamic parameters involved in cellular condensate transitioning from super-resolved images. Therefore, I am convinced that this manuscript will be of great interest to the broad scientific public interested in the mechanism involved in the self-organization of molecular condensates.